Drop-in biofuel production using fatty acid photodecarboxylase from Chlorella variabilis in the oleaginous yeast Yarrowia lipolytica

Abstract

Background Oleaginous yeasts are potent hosts for the renewable production of lipids and harbor great potential for derived products, such as biofuels. Several promising processes have been described that produce hydrocarbon drop-in biofuels based on fatty acid decarboxylation and fatty aldehyde decarbonylation. Unfortunately, besides fatty aldehyde toxicity and high reactivity, the most investigated enzyme, aldehyde-deformylating oxygenase, shows unfavorable catalytic properties which hindered high yields in previous metabolic engineering approaches. Results To demonstrate an alternative alkane production pathway for oleaginous yeasts, we describe the production of diesel-like, odd-chain alkanes and alkenes, by heterologously expressing a recently discovered light-driven oxidase from Chlorella variabilis (CvFAP) in Yarrowia lipolytica. Initial experiments showed that only strains engineered to have an increased pool of free fatty acids were susceptible to sufficient decarboxylation. Providing these strains with glucose and light in a synthetic medium resulted in titers of 10.9 mg/L of hydrocarbons. Using custom 3D printed labware for lighting bioreactors, and an automated pulsed glycerol fed-batch strategy, intracellular titers of 58.7 mg/L were achieved. The production of odd-numbered alkanes and alkenes with a length of 17 and 15 carbons shown in previous studies could be confirmed. Conclusions Oleaginous yeasts such as Yarrowia lipolytica can transform renewable resources such as glycerol into fatty acids and lipids. By heterologously expressing a fatty acid photodecarboxylase from the algae Chlorella variabilis hydrocarbons were produced in several scales from microwell plate to 400 mL bioreactors. The lighting turned out to be a crucial factor in terms of growth and hydrocarbon production, therefore, the evaluation of different conditions was an important step towards a tailor-made process. In general, the developed bioprocess shows a route to the renewable production of hydrocarbons for a variety of applications ranging from being substrates for further enzymatic or chemical modification or as a drop-in biofuel blend. Electronic supplementary material The online version of this article (10.1186/s13068-019-1542-4) contains supplementary material, which is available to authorized users.

Engineering nature for gaseous hydrocarbon production

“…In addition, this enzyme requires the co-expression of ferredoxin to supply an electron transfer system. For bio-alkane production, upregulation and subsequent accumulation of fatty aldehyde precursors is problematic due to the reactive and toxic nature of these compounds [9,20,44,45]. In spite of this, multiple studies have demonstrated successful moderate production of gaseous and non-gaseous hydrocarbons in Escherichia coli and other microorganisms [5, 8-10, 20, 46].…”

Bacillus subtilisspore surface display of photodecarboxylase for the transformation of lipids to hydrocarbons

“…Detection of HCs was performed by gas chromatography as described by [14] Rate 20, final temp. 300 °C, hold 5 min.The analytical GC grade standards undecane, tridecane, pentadecane, heptadecane and the C8-C20 alkane standard solution were purchased at Sigma-Aldrich.…”

Evaluation of a Yarrowia lipolytica Strain Collection for Its Lipid and Carotenoid Production Capabilities

“…In order to transfer the genes required for carotenoid production, the prototrophic strains had to be made auxotrophic for uracil in order to enable application of the in yeast commonly applied URA3 marker gene for selection. Disruption of the native, chromosomal URA3 gene in all strains of this study was performed using a CRISPR-Cas9 system harboring a hygromycin marker cassette, [8] CRISPRyl-Hyg-URA3. Transformation, counterselection on 5-fluoroorotic acid and verification by sequencing are de-scribed in the above mentioned study.…”

“…Yeast strains were cultivated in YPD medium and 180 rpm in Cellstar 24-well cell culture plates (Greiner bio-one, Austria) using a custom microwell plate-holder described in ref. [8]. For the determination of optical density, a PHERAstar FSX microplate reader (BMG Labtech, Germany) was used.…”